Fluorescent molecular probes that can label, detect, or image specific proteins serve as a powerful tool for developing in-vitro proteomic assays, for identifying disease biomarkers, as well as for tracking proteins in their native environments. Ideally, such probes should act as ‘turn-on’ fluorescent molecular sensors, which do not generate any background signal in the absence of the bioanalyte, but emit very strongly in the presence of the protein target. In practice, however, developing fluorescent molecular switches that can recognize their target proteins with high affinity, selectivity, and sensitivity is challenging. Obtaining highly selective sensors is complicated by the fact that many protein groups, which can be targeted by small-molecule-based sensors, possess well-defined recognition sites that are conserved among structurally similar isoforms of the same family. High sensitivity is also difficult to achieve because common fluorescence signaling mechanisms, such as photo-induced electron transfer (PET), charge transfer (CT), or fluorescence resonance energy transfer (FRET) often lead to a background emission signal by the unbound sensors. Consequently, an excess of protein is generally required to obtain a sufficient fluorescence response. Finally, a limitation of many molecular sensors, when compared with the corresponding antibodies or aptamers, is that they bind their target with lower affinities, which prevents them from detecting proteins at low concentrations.
Asymmetrical cyanine dyes constitute a unique class of fluorescent molecular sensors whose activation does not involve FRET, ICT, or PET processes. Instead, their fluorescence emission is turned on upon restriction of their torsional motion. For example, the emission of Thiazole Orange (TO) is quenched due to excited state twisting of benzothiazole and quinoline rings around the methine bridge, which leads to a non-radiative decay. Binding to DNA or peptide aptamers, or interchelation into DNA duplexes restricts this torsional motion and leads to an enhanced fluorescence signal.
Human soluble GSTs can be mainly subdivided into 7 classes, namely, α (A), μ (M), π (P), θ (T), σ (S), κ (K), and ω (O), which differ in their electrophilic substrate preferences as well as in their tissue distribution. Comparative analysis of GST expression in normal and diseased tissues or serum has shown a clear correlation between their expression profiles and disease states. For example, abnormal tissue expression of GST α (A) has been associated with an increased risk for colorectal cancer, ovarian cancer, and clear cell renal cell carcinoma, μ (M) class expression alteration was detected in cases of lung, colon, and bladder cancer, whereas the π (P) class isozymes are overexpressed in the majority of human tumors. Moreover, several metabolic conditions led to excretion of GST proteins into urine or the blood circulation. For instance, the presence of GST-A1 in urine or in blood plasma is an early biomarker for hepatocellular damage, whereas elevated serum levels of GST-P1 is an indicator of various cancers (breast, lung and gastric cancers). GST-A1 is an indicator for colorectal, prostate, breast and lung cancers. GST-A2 is an indicator for prostate and lung cancers. GST-M1 is an indicator for prostate and breast cancers. An issue of high importance is distinguishing between combinations of several GST subtypes in urine. For example, measurements of GST-A and GST-P in urine provide information about the site of renal tubular injury. In addition, a combination of plasma α and π levels was proposed as a tool to predict and monitor graft failure or regeneration following living donor liver transplantation.
GSTs are also commonly used as fusion proteins, which facilitate the purification of GST-labeled protein with a GSH column. Fluorescent molecular sensors for GSTs could therefore be used for detecting GST-labeled proteins in living cells.
Acetylcholinesterase, an important biomarker for the Alzheimer's disease, is a hydrolase that hydrolyzes the neurotransmitter acetylcholine and regulates the concentration of this transmitter at the synapse. AChE is found at mainly neuromuscular junctions and cholinergic brain synapses, where its activity serves to terminate synaptic transmission. It is the primary target of inhibition by organophosphorus compounds such as nerve agents and pesticides.
Avidin/streptavidin-biotin system is a powerful tool in biological sciences. The strength and specificity of the avidinistreptavidin-biotin complex, is exploited by researches for their use as probes and affinity matrices in numerous research projects and biologica assays including western blot, ELISA, ELISPOT and pull-down assays. Avidin and Streptavidin are used in applications ranging from research and diagnostics to medical devices and pharmaceuticals.
Fibroblast Growth Factors (FGFs) are a family comprising 22 heparin-binding proteins whose over-expression is associated with different types of cancers. Fluorescent molecular sensors, specific particular FGFs, could therefore facilitate identifying medicinally relevant samples involving different combinations or concentrations of FGFs.
Estrogen Receptors (ERα) have been mainly implicated in the development and progression of breast cancer, where much research has focused on identifying alterations within the coding sequence of these receptors in clinical samples. Mutations within ERα have been identified in several different diseases, indicating that the most common technique for determining tumor ER status, namely, immunohistochemical assays or ligand binding assays, might not be efficient for identifying ERs with abnormal ligand binding capacity or reduced functionality. Therefore, fluorescent molecular sensors, specific for ERs, might serve as an additional tool for characterizing ER biomarkers.
Matrix Metalloproteases (MMPs) family of enzymes comprising more than 20 zinc-dependent endopeptidases that share a similar, zinc-dependent binding site, and are capable of degrading virtually every component of the extracellular matrix (ECM). These isozymes can be divided into several subgroups, based on their structures or preferential substrates, which include, among others, collagen, gelatin, and various extracellular matrix proteins.
Owing to their role in tumor growth, metastasis, and angiogenesis, MMPs are considered as important therapeutic targets for treating human cancers. In addition, high levels of members of the MMP family in serum, urine, or tissue have been identified in a variety of human cancers, including breast, pancreatic, bladder, colorectal, ovarian, and prostate cancer (for example, MMP-1 is identified in breast cancer, lung cancer and colorectal cancer; MMP-2 is identified in pancreas cancer, bladder cancer, colorectal cancer, ovarian cancer, prostate cancer and brain cancer; MMP-7 is identified in pancreas cancer, lung cancer and colorectal cancer; MMP-9 is identified in breast cancer, pancreas cancer, bladder cancer, lung cancer, colorectal cancer, ovarian cancer, prostate cancer and brain cancer). Thus, MMPs are considered to be promising biomarkers for different cancers, both for diagnostic and prognostic purposes. Glutathione S-Transferases (GSTs) are a family of widely distributed enzymes that play a role in cell detoxification by catalyzing the conjugation of γ-L-glutamyl-L-cysteinylglycine (gluthation) to a broad range of electrophilic endotoxines and xenobiotics that are subsequently excreted from the cell. This activity is a crucial part of a self-defense mechanism that protects the organism from toxic and sometimes carcinogenic species.
Genetically encoded fluorescent proteins (FPs) have revolutionized the study of biology by allowing one to track protein expression and localizations in living cells at spatial and temporal resolution. This method, however, involves the use of very large protein tags that can interfere with the normal function of the labeled protein. Over the last few years, it has been demonstrated that this problem can be circumvented by expressing the proteins with a very short peptide sequence to which a small fluorescent molecular sensor, termed “genetically-targeted sensors” can attach. Sensors that can bind to an oligohistidine sequence (i.e. His-tag) with high affinity and can be applied for labeling and detecting a wide range of His-tagged proteins in living cells.
The above examples not only stress the importance of developing methods for high-throughput protein analysis in biological fluids but also highlight GSTs, AChE, FGFs, ER and MTPs as potential biomarkers for detecting early stages of various diseases, including cancer and Alzheimer.
Surprisingly, despite the remarkable analytical power of fluorescent molecular sensors and their success in detecting various biomolecules and ions in aqueous solutions, the development of ‘turn-on’ fluorescent molecular switches for proteins, which do not rely on enzymatic reactions, has been relatively scarce.
This invention shows that the conversion of a known intercalating dye (e.g., Thiazole Orange) into a bivalent protein binder could lead to the realization of a novel class of fluorescent molecular sensors that detect proteins, including individual protein isoforms, with high affinity, selectivity, and excellent signal-to-noise (S/N) ratio. The feasibility of the approach is demonstrated with monomolecular sensors that light-up in the presence of various proteins (e.g. glutathione-s-transferase (GST), avidin (Av), acetylcholinesterase (AChE) etc.) at low concentrations and with minimal background signal. Such sensors are also able to respond differently to the surfaces of distinct protein isoforms, which circumvents the challenge of developing a highly selective binder for each family member. This property, thus, opens up new possibilities for using sensors appended with broad-spectrum protein binders in order to obtain isoform-specific detection.
Therefore, and given that about 30% of human proteins are homodimers, the protein sensors presented herein are expected to contribute to the development of ‘turn-on’ fluorescent molecular switches for proteins, which do not rely on enzymatic reactions, by affording a novel methodology for selective and sensitive detection of a wide range of different proteins and protein isoforms.